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Abstract:

The present disclosure provides a method of modifying cells in order to
enhance lentiviral titers, cell lines that are modified and modifying
reagents. By mediating individual genes and combination thereof,
lentiviral titers may be increased.

Claims:

1. A method of enhancing lentiviral titers comprising modulating
expression of a host gene in a packaging cell, wherein the host gene is
Tetherin.

2. The method of claim 1, wherein the packaging cell is an HEK-293 cell.

3. The method of claim 1, wherein said modulating comprises introduction
of an siRNA, wherein said siRNA comprises an antisense strand and a sense
strand, wherein the antisense strand is complementary to a region of
Tetherin.

6. A method of enhancing lentiviral titers comprising modulating
expression of a plurality of host genes in a packaging cell, wherein the
plurality of host genes comprise Tetherin and at least one gene selected
from the group consisting of Dicer, DGCR8, TSG101 and Anx2.

7. The method of claim 6, wherein the at least one gene is Dicer.

8. The method of claim 6, wherein the at least one gene is DGCR8.

9. The method of claim 8 further comprising modulating expression of
Dicer.

10. The method of claim 6, wherein the at least one gene is TSG101.

11. The method of claim 6, wherein the at least one gene is Anx2.

12. The method of claim 11 further comprising modulating expression of
TSG101.

Description:

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application is a divisional of U.S. Ser. No. 13/222,509, filed
Aug. 31, 2011, pending, which claims the benefit of the filing date of
U.S. Provisional Application Ser. No. 61/388,338, filed Sep. 30, 2010,
the entire disclosures of which are incorporated by reference as if set
forth fully herein.

FIELD OF INVENTION

[0002] The present invention relates to lentiviruses

BACKGROUND OF INVENTION

[0003] Lentiviruses (LVs) designed to deliver transgenic cargo (e.g., a
protein coding sequence or an shRNA) are valuable tools in basic
research, bioproduction, and therapeutic delivery. In order to generate
these valuable reagents, researchers generally introduce a transfer
vector (encoding the desired transgenic viral genome to be packaged)
along with one or more packaging vectors (that produce essential viral
proteins) into an appropriate packaging cell line (e.g. HEK 293 cells).
The inventors have recognized that the current set of reagents and
protocols is highly inefficient and that new methods are needed to
develop sufficient quantities of viral particles. For this reason, the
following invention describes novel and non-obvious sets of reagents and
processes for greatly enhancing LV production.

SUMMARY

[0004] The present invention provides modified packaging cell lines and
methods for efficient production of lentiviral particles. Specifically,
in some embodiments the cells have been altered in such a way as to
knockout or knockdown one or more host encoded genes that negatively
affect lentiviral vector particle formation in the packaging cell line.
Alternatively or in addition to the aforementioned alterations, the cells
have been modified to over-express one or more host encoded genes that
facilitate viral particle formation and release. Host genes that can be
targeted (for knockout or knockdown) to enhance LV particle formation and
release include, but are not limited to, Dicer, Drosha, DGCR8, Hrs, and
Tetherin. Host genes that can be over-expressed to enhance LV particle
formation and release include, but are not limited to, Annexin2 and
Tsg101. The present invention describes individual knockouts (or
knockdowns) and enhancements, as well as combinations of knockouts (or
knockdowns) and/or enhancements, as well as the effects that modulating
these genes (singularly or in combination) have on viral production.

BRIEF DESCRIPTION OF FIGURES

[0005]FIG. 1A shows a schematic of a typical lentiviral transfer vector,
which includes for instance, an open reading frame (ORF), an IRES
(internal ribosomal entry sequence) sequence for cap-independent
translation, a reporter construct to identify cells that have been
transduced, as well as a 2A peptide separating the reporter construct
from a selectable marker (e.g. puromycin). Alternative designs that, for
instance, drive the expression of an RNAi silencing reagent (e.g., an
shRNA) are envisioned. FIG. 1B is a simplified schematic of the steps
involved in creating a lentiviral particle. FIG. 1c is a simplified
schematic of the lentiviral lifecycle including cellular entry, reverse
transcription of the viral genome from RNA→DNA, transport into the
nucleus, and integration into the host genome.

[0006]FIG. 2 is a graph showing the effects of each of Dicer, Drosha, and
DGCR8 knockdown on lentiviral titers. NTC represents a non-targeting
control.

[0007]FIG. 3 is a graph showing the effects that four separate siRNAs
targeting DGCR8 have on LV titer.

[0008]FIG. 4 is a graph showing the effects of DGCR8 knockdown on LV
titers when the siRNA, packaging vectors, and transfer vector, are
simultaneously introduced into cells.

[0009]FIG. 5 is a graph showing the effects that knockdown of Hrs or
Tetherin have on LV titers.

[0010]FIG. 6 is a graph showing the effects that over-expression of
Tsg101 or Annexin2 (Anx2) have on LV titers. MOI=multiplicity of
infection.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0011] The present disclosure will now be described in connection with
preferred embodiments. These embodiments are presented to aid in an
understanding of the present disclosure and are not intended, and should
not be construed, to limit the disclosure in any way. All alternatives,
modifications and equivalents that may become apparent to those of
ordinary skill upon reading this disclosure are included within the
spirit and scope of the present disclosure.

[0012] The present disclosure is directed to compositions and methods for
generating lentiviral particles. Through the use of the present
disclosure, modified cells and derivatives thereof may be constructed to
enhance lentiviral production.

[0013] Lentiviruses can be used to transfer genetic material (e.g.,
protein coding genes, miRNAs, and shRNAs) into cells. To achieve this, a
lentiviral transfer vector or plasmid comprising, consisting essentially
of or consisting of DNA encoding the necessary elements for packaging
into a lentiviral particle (see FIG. 1A) is transfected into cells along
with helper vectors (see FIG. 1B). In the packaging cell, the information
associated with the (DNA) transfer vector is transferred to a viral RNA
genome, which is then packaged into a lentiviral particle that buds from
the cell into the surrounding media. Lentiviral particles created by this
method are then capable of infecting or "transducing" target cells (FIG.
1C). During the course of transduction, the information associated with
the viral RNA genome is transferred into DNA that is then delivered to
the nucleus where it is stably integrated into the host chromosome. In
this fashion, long-term expression of the genetic payload can be
achieved.

[0014] A critical parameter of lentiviral production is viral titer. In
this instance, viral titer is defined as the number of infectious
particles per volume, and lentiviral packaging protocols that produce
higher titers greatly facilitate research and developmental studies.
Historically, lentiviral titers are low (104-107 infectious
viral particles per ml), and previous studies have identified several
factors (including the size of the transfer vector, the method of
introducing the transfer and packaging vectors into the packaging cells,
and the ratio of transfer vectors to packaging vectors) that contribute
to the viral titer. Still, even when all of these elements have been
optimized, overall viral titers are still lower than those obtained with
other viral production systems (e.g., adenovirus, >1010 particles
per ml).

[0015] In the course of the work presented here, the inventors have
discovered that modulating the expression of a combination of host
(cellular) genes strongly enhances lentiviral production. Specifically,
modulation (which includes both up and down regulation of specific host
genes) has been shown to increase overall viral titers by ten-fold or
more. Thus, according to the first embodiment, the present disclosure is
directed to a cell line in which a combination of host genes is modulated
to enhance production of virus, or virus-like particles (VLPs) and a
method of producing high viral titers using such cell line.

[0016] It should be noted that while many of the genes have been shown to
alter viral titer, other sets of (host) genes may be modulated to enhance
the infectivity of the resulting viral particles. Under these instances,
the overall numbers of viral titers (per se) may not change, but the
ability of a given particle to transduce a cell would increase. In some
embodiments, the present invention is directed to cell lines, in which a
first gene has been modulated to enhance production of viruses or
virus-like particles, and a second gene has been modulated to enhance the
infectivity of the resulting viral particle. In various embodiments, the
present invention is also directed to methods for making or using these
cell lines.

[0017] A variety of cells/cell types including primary cells or cells
derived from a cell line can be used in the invention. In cases where the
cells are derived from a cell line, the cell line can be a transgenic
cell line specifically modified to produce e.g., LV particles. For
instance, the cell line can represent a minimal packaging cell line that
has been modified to express (in a constitutive or inducible manner) one
or more of the following genes including (but not limited to) gag-pol,
tat, or rev. In addition, the cells can be mammalian or non-mammalian in
origin and be adherent or non-adherent in nature. Preferably, the cells
of the invention are adherent cells. More preferably, the cells are
derived from human embryonic kidney cells with examples of desired cells
including but not limited to HEK-293 cells (ATCC No. CRL-1573) and HEK
293T cells (ATCC No CRL-11268). Other cell lines that can be used in the
present invention are available from ATCC.

[0018] As stated above, the inventors have identified combinations of
host-encoded genes that when modulated (i.e., up- or down-regulation of
function) enhance lentiviral production. In the context of the invention,
host-encoded gene products that inhibit LV production are genes that are
targeted for suppression in order to enhance LV titers. Conversely,
host-encoded gene products that facilitate LV production are genes whose
function is bolstered to enhance LV titers. The host-encoded genes that
can be modulated can include both protein encoding genes and non-protein
encoding genes (e.g., miRNAs).

[0019] With respect to gene suppression, complete suppression of the
target gene function is unnecessary to facilitate LV production, and as
such, technologies that knockdown or knockout function are compatible
with the invention. For this reason, there are a variety of technologies
that can be employed to curb gene function. These include, but are not
limited to, knockdown or knockout of the target gene at the genome-level
(e.g., by zinc-finger-based technologies, transposon/insertion-based
mutatgenesis), suppression of gene transcription (e.g., by epigenetic
mechanisms), degradation and/or suppression of translation of the target
transcript (e.g., via RNAi-based technologies), or suppression of protein
function by any number of mechanisms including but not limited to
altering post-translational modification patterns, exposure to a known
modulator protein(s), a regulatory peptide, or a small molecule, that
alters the function of the host target protein. In one preferred method,
gene function is suppressed using RNAi technology. Under these
conditions, knockdown can be transient (e.g., mediated by siRNA),
constitutive (e.g., mediated by stable expression of an shRNA from a
plasmid or integrated expression construct), or regulated (e.g.,
ligand-mediated shRNA expression). In cases where the strategy involves
e.g., stable knockdown, the inventors envision the development of
modified cell lines to achieve this goal. Described RNAi reagents can be
delivered to cells by any number of mechanisms including, but not limited
to, transfection (using lipids, peptide-based delivery reagents, or
alike), mechanical methods (e.g., electroporation), passive delivery, or
transduction.

[0020] RNAi can be accomplished in mammals by using siRNA. When using
siRNA, one may, for example, use duplexes that have 18-30 bases pairs,
19-30 base pairs, 19-23 base pairs or 19 base pairs and either no
overhangs or one or more overhangs at the 3' or 5' ends of the sense or
antisense strands. Furthermore, the siRNA may be modified or unmodified.
Examples of modifications include those described in U.S. Pat. No.
7,595,387, issued Sep. 29, 2009 and U.S. Pat. Pub. 2009/0209626,
published Aug. 20, 2009, the entire disclosures of which are both
incorporated by reference as if set forth fully herein. These
modifications may include one or more sense strand modifications and/or
one or more antisense strand modifications. The sense strand
modifications may for example be one or more of 2'-O-methyl modifications
on nucleotides 1 and 2 (counting from the 5' end of the strand),
2'-O-methyl modifications on all Cs and Us and cholesterol conjugated to
the 3' terminus using a C5 linker. The antisense strand modifications may
be one or more of a 5' phosphate, 2' F on all Cs and Us, a two nucleotide
(UU) overhang on the 3' terminus and phosphorothioate internucleotide
modifications between the two nucleotides of the overhang and between the
first (3' most) nucleotide of the duplex and the first nucleotide of the
overhang. By convention, when referring to RNA unmodified nucleotides are
presumed to have OH groups at their 2' positions.

[0021] In addition, mismatches at positions 6, 13, and 19 of the sense
strand may be incorporated into molecules. In all cases, mismatches
between the two strands of the siRNA are achieved by changing the
nucleotide of the sense strand to have identity with the base (on the
antisense strand) that typically pairs with that position. Thus, for
instance, if the sense-antisense pair at sense strand position 6 is
normally U-A, then the mismatch will be introduced by converting the pair
to A-A. Similarly, if the sense-antisense pair at sense strand position 6
is G-C, then the mismatch will be C-C. In this way, a mismatch is
incorporated into the duplex, but the antisense strand remains the
complement of the intended target. In some embodiments, there are no
mismatches.

[0022] Examples of duplexes used in connection with the present invention
may contain a sense sequence that is delineated in Table I and/or an
antisense sequence that is the complement of a sequence delineated in
Table I. The duplexes may have none or any one or more of the
aforementioned modifications, including a mismatch as described above.

[0023] Multiple different technologies can also be used to enhance or
up-regulate gene function. These can be used alone or in combination and
include but are not limited to: (1) enhancing transcription by e.g.,
epigenetic means or by modulating gene copy number; (2) enhancing gene
translation (e.g., by limiting translation attenuation or transcript
degradation by miRNAs); and/or (3) modulating protein function by any
number of means including but not limited to creating a constitutively
active form of the protein or "evolving" protein function by in vitro
mechanisms, altering post-translational modification patterns that
regulate protein function (e.g., elimination of phosphorylation sites),
or enhancing protein function using e.g., small molecules or peptides. In
one preferred method, gene function is enhanced using over-expression
constructs. Over-expression of a desired protein can be achieved by a
number of means including, but not limited to introduction of an
expression construct (e.g., a plasmid or viral construct) comprising a
constitutive or regulated promoter sequence operationally linked to an
ORF or cDNA sequence. Additionally, one may develop stable cell lines
that, for instance, exhibit enhanced or up-regulated function of a
desired gene.

[0024] Host genes identified by the invention can be suppressed
singularly, or in combination with other gene functions that are
suppressed, and/or in combination with still other gene functions that
are enhanced or up-regulated. Examples of host genes that can be
suppressed include, but are not limited to, Dicer, Drosha, DGCR8, Hrs,
Tetherin, CDK13, TRIM-5alpha, PRMT6, as well as miR-29a, b, and c.
Similarly, host-encoded gene functions can be enhanced/up-regulated
singularly, in combination with other gene functions that are enhanced or
up-regulated, and/or in combination with gene functions that are
suppressed. Examples of host genes that can be enhanced include, but are
not limited to, Annexin2, Tsg101, DBR1, RNA Helicase A, SOCS1,
Cyclophillin A and TRBP.

[0025] The reagents and processes of the invention can be applied to
enhance the production of a number of viruses including but not limited
to a wide range of lentiviruses as well as retroviruses. As such, the
invention has utility in multiple fields including those associated with
industry and therapeutics. In industry, the invention can be used to
greatly enhance the production of e.g., lentiviral particles carrying
e.g., cDNA, ORF, or shRNA payloads, thereby reducing the costs associated
with production of these important reagents for academic, industrial, and
governmental researchers. Similarly, in therapeutics, the invention can
be used to greatly enhance the production of lentiviral particles
carrying cDNAs, ORFs, and/or shRNAs used in the treatment of both human
and non-human diseases. Alternatively, the invention can be used to
greatly enhance the production of e.g., lentiviral particles used in
e.g., vaccines for HIV, FIV, and other lenti- or retroviruses.

[0026] As noted above, suppression of genes that inhibit lentiviral titers
may be complete or partial. For example, suppression may be from 10% (90%
expression) to 100% (complete suppression) relative to a normal
expression, or from 5% to 10% suppression, or from 10% to 20%
suppression, or from 30% to 40% suppression, or from 40% to 50%
suppression, or from 50% to 60% suppression, or from 60% to 70%
suppression, or from 70% to 80% suppression, or from 80% to 90%
suppression. Similarly, enhancement of genes that facilitate expression
of lentiviral titers may be designed to enhance expression by greater
than 5%, greater than 10%, greater than 20%, greater than 30%, greater
than 40%, greater than 50%, greater than 60%, greater than 70%, greater
than 80%, greater than 100%, greater than 150% or greater than 200%.

[0027] When carrying out the methods of the present invention one may
first select a cell type or cell line that is compatible with 1) standard
transfection procedures (lipid- or calcium phosphate mediated), and 2)
the replication, packaging, and release of lentiviral viruses. Examples
of cell lines that would be compatible with the invention include, for
instance, HEK 293T cells.

[0028] In addition to selecting a cell line, it is necessary to select the
necessary genetic elements (plasmids, vectors) needed to generate a
lentiviral particle. Both the number of functions (and therefore the
number of vectors/plasmids) required to generate a lentiviral particle
can vary. In one preferred method, the materials would include a
lentiviral transfer vector/plasmid that contains the necessary genetic
elements 1) to be packaged into a lentiviral particle, and 2) to infect a
target cell. For example, the transfer vector may include a 5' and 3'
LTR, a cPPT site, and a Psi site. In addition, the materials would
include one or more accessory packaging plasmids that provide additional
functions needed for packaging e.g., the transfer vector/plasmid into a
viral particle. Functions encoded by the one or more packaging plasmids
may include an envelope protein (env) expression cassette and a
polymerase protein expression cassette. It should be noted that while the
necessary functions described above are delivered to cells via plasmid or
vector expression constructs, it is also possible that a transgenic cell
line can be generated that expresses one or more of the needed functions
in the packaging cell line in a constitutive or regulated fashion.

[0029] In order to generate a lentiviral preparation of high titer, one
would begin by plating the cell line described above in e.g. a tissue
culture plate with the appropriate media. After a sufficient period of
time (e.g., overnight), the researcher may introduce an siRNA sequence
(or sequences) into the cells or cause one or more siRNA or shRNA
sequences to be generated in the cell by a vector that is introduced or
already present. Alternatively, the researcher may treat the cell with a
reagent (e.g., a small molecule, an antisense molecule) that, like the
siRNA, targets the gene of interest. Following a period of incubation
that is sufficient for the agent (e.g., siRNA) to knockdown or inhibit
the target gene of interest, the cells would be transfected with the
previously described genetic elements (plasmids, vectors) needed to
generate a lentiviral particle. Methods for introducing a plasmid or
plasmids into cells are well recognized in the art and include
electroporation, lipid-mediated transfection, and more. Following this
procedure, cells would be cultured using standard tissue culture
practices. At an appropriate period of time (generally 24-96 hrs
post-transfection) the culture supernatant containing the high titer
virus can be collected and used immediately.

EXAMPLES

Example I

Effects of Knockdown of Genes Associated with the RNAi Pathway on
Lentiviral Titer

[0031] Twenty-four hours post-transfection (Day 3) cells were
co-transfected with a pool of plasmids that included a lentiviral
transfer vector (6 micrograms/well) and accessory packaging vectors
(TransLenti Packaging Mix, Thermo Fisher Scientific, Open Biosystems
Products) using calcium phosphate precipitation. Cell culture media was
replaced 18 hours post-transfection and viral supernatants were collected
48 hours later. Viral titer was then determined by transduction, which
involved infecting 293T cells with dilutions of the viral supernatant and
measuring the number of cells expressing the viral-associated GFP
reporter vector. All experiments were done in triplicate and results were
normalized to a non-targeting control siRNA (NTC cat. no. D-001210-01-05,
Thermo Fisher Scientific, Dharmacon Products).

[0032] The results of these studies are shown in FIG. 2. All three
knockdowns were successful. The siRNA-mediated knockdown of Drosha
enhanced overall viral titer by about 1.5 times. RNA mediated knockdown
of Dicer enhanced overall titers by over 1.5 times while RNAi mediated
the knockdown of DGCR8 increased titers by over two-fold.

[0033] As a follow up to these experiments, the researchers assessed
whether all of the siRNA in the pool of four siRNAs performed
equivalently. Protocols were identical to those described previously with
the exception that individual siRNA were introduced into cells at
concentrations of 25 nM rather than pools. All experiments were performed
in duplicate. As shown in FIG. 3, three out of the four siRNA present in
the original pool increased viral titers. While siRNA duplex #3 failed to
affect overall titers, duplex #4 increased titers by roughly two-fold
while duplexes #1 and #2 increased viral titer by 3.5-4 fold.
Demonstration that multiple siRNA induce similar phenotypes validates
DGCR8 as a host-encoded target that (when silenced) greatly enhances
lentiviral titers.

[0034] In a separate experiment, the inventors tested whether simultaneous
transfection of DGCR8 siRNA together with 1) LV transfer vectors, and 2)
LV packaging vectors led to an increase in lentiviral titers. As was the
case in previous experiments, packaging cells were seeded and cultured
overnight. On Day 2, the pools of siRNA targeting DGCR8 were
co-transfected into cells along with the LV transfer and packaging
vectors using calcium phosphate precipitation. Three days
post-transfection (Day 5), viral supernatants were collected and titers
were determined as previously described. As shown in FIG. 4, simultaneous
introduction of the DGCR8 siRNAs along with the packaging mixture
provided significant increases in viral titer.

Example II

Effects of Knockdown of Hrs and Tetherin on Lentiviral Titers

[0035] Using the original protocol, the researchers investigated the
effects that siRNA-mediated knockdown (KD) of Hrs and Tetherin (Thermo
Fisher Scientific, Dharmacon Products, cat. no. M-011817-00-0005
(Tetherin) and M-016835-00-0005 (Hrs)) would have on lentiviral titers.
As shown in FIG. 5, KD of Hrs provided only modest increases in viral
titers (<1.5× effects). In contrast, KD of Tetherin induced
significant increases in viral titer (greater than two fold, p<0.05).

Example III

Effects of Over-expression of Annexin2 and Tsg101 on Lentiviral Titers

[0036] To test the effects that over-expression of Annexin2 has on
lentiviral titers, lentiviral particles capable of expressing the
Annexin2 or Tsg101 open reading frame (ORF) were transduced into HEK293T
cells at an MOI of either 0.3 or 3.0 (LentiORF, Thermo Fisher Scientific,
Open Biosystems Products cat. no. PLOHS--100003638 (Annexin 2) and
PLOHS--100005422 (Tsg101)). Subsequently, cultures were grown in the
presence of blasticidin (10 ug/ml, 2 week) to select for stable
integrants. Cells were then expanded for 1 week in the absence of
blasticidin, seeded at a density of 1.2×106 cells per well in
a 6 well plate, and then transfected with a transfer vector/Trans-Lenti
packaging mixture (available from Thermo Scientific).

[0037] The results of these studies are shown in FIG. 6 which shows that
over-expression of TSG101, as well as Annexin 2 increase in viral titers.

[0038] The studies described above have identified multiple host-encoded
genes that when modulated individually (down- or up-regulated) can have
significant effects on lentiviral vector particle production.
Accordingly, based on the work of the inventors and described herein it
is predicted that modulating a set of genes (particularly a combination
of genes with different functions, e.g. a gene involved in RNAi pathway
and another gene involved in viral packaging) simultaneously will yield
even higher LV titers than modulating any individual gene. Thus, for
example, knockdown of DGCR8 could be accompanied by knockdown of Hrs or
Tetherin, or over-expression of Tsg101 or Annexin2. Alternatively,
knockdown of Hrs could be accompanied by knockdown of Tetherin or
over-expression of Tsg101 or Annexin 2. In yet a further permutation,
down-regulation of Tetherin could be paired with over-expression of
Tsg101 or Annexin2. In still another permutation, over-expression of
Tsg101 could be paired with over-expression of Annexin2.

[0039] Further, additional increases in LV titers can be achieved by
modulating three or more genes simultaneously. Thus, for example, three
separate genes, DGCR8, Hrs, and Tetherin, could be knocked-down
simultaneously to further enhance viral titers. Alternatively, other
combinations of the genes (e.g., DGCR8+Hrs+Anx2) identified by these
studies could be modulated simultaneously to further enhance LV
production. Lastly, all of the genes identified in these studies can be
modulated simultaneously to enhance LV production. Stable cell lines can
be generated with any of the aforementioned modulating genes.